EP3105255A1 - Glucosyltransferase enzymes for production of glucan polymers - Google Patents
Glucosyltransferase enzymes for production of glucan polymersInfo
- Publication number
- EP3105255A1 EP3105255A1 EP15705889.2A EP15705889A EP3105255A1 EP 3105255 A1 EP3105255 A1 EP 3105255A1 EP 15705889 A EP15705889 A EP 15705889A EP 3105255 A1 EP3105255 A1 EP 3105255A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- glucan
- poly alpha
- alpha
- seq
- linkages
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 238000000034 method Methods 0.000 claims abstract description 51
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- 125000003275 alpha amino acid group Chemical group 0.000 claims description 29
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- 229940006486 zinc cation Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 0.000 description 1
- XJUNLJFOHNHSAR-UHFFFAOYSA-J zirconium(4+);dicarbonate Chemical compound [Zr+4].[O-]C([O-])=O.[O-]C([O-])=O XJUNLJFOHNHSAR-UHFFFAOYSA-J 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
Definitions
- This invention is in the field of polysaccharides and polysaccharide derivatives. Specifically, this invention pertains to certain poly alpha-1 ,3-1 ,6- glucans, glucosyltransferase enzymes that synthesize these glucans, ethers of these glucans, and use of such ethers as viscosity modifiers.
- polysaccharide is poly alpha-1 ,3-glucan, a glucan polymer characterized by having alpha-1 ,3- glycosidic linkages.
- Poly alpha-1 ,3-glucan has been isolated by contacting an aqueous solution of sucrose with a glucosyltransferase (gtf) enzyme isolated from
- U.S. Patent 7,000,000 disclosed the preparation of a polysaccharide fiber using an S. salivarius gtfJ enzyme. At least 50% of the hexose units within the polymer of this fiber were linked via alpha-1 ,3-glycosidic linkages. The disclosed polymer formed a liquid crystalline solution when it was dissolved above a critical concentration in a solvent or in a mixture comprising a solvent. From this solution continuous, strong, cotton-like fibers, highly suitable for use in textiles, were spun and used.
- glucan polysaccharides and derivatives thereof are desirable given their potential utility in various applications. It is also desirable to identify glucosyltransferase enzymes that can synthesize new glucan polysaccharides, especially those with mixed glycosidic linkages and high molecular weight.
- the invention concerns a reaction solution comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha- 1 ,3-1 ,6-glucan.
- the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- glucosyltransferase enzyme has a weight average degree of polymerization (DPw) of at least 1000.
- At least 60% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan synthesized by the glucosyltransferase enzyme are alpha- 1 ,6 linkages.
- the DP W of the poly alpha-1 ,3-1 ,6-glucan synthesized by the glucosyltransferase enzyme is at least 10000.
- the glucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the invention concerns a method for producing poly alpha-1 ,3-1 ,6-glucan comprising the step of contacting at least water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1 ,3-1 ,6- glucan.
- the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the poly alpha-1 ,3-1 ,6-glucan produced in this method can optionally be isolated.
- At least 30% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan synthesized by the glucosyltransferase enzyme in the method are alpha-1 , 3 linkages
- at least 30% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan are alpha-1 ,6 linkages
- the poly alpha-1 ,3-1 ,6- glucan has a DP W of at least 1000.
- At least 60% of the glycosidic linkages of the poly alpha-1 , 3-1 , 6-glucan synthesized by the glucosyltransferase enzyme in the method are alpha-1 , 6 linkages.
- the DP W of poly alpha-1 , 3-1 , 6-glucan synthesized by the glucosyltransferase enzyme in the method is at least 10000.
- invention or “disclosed invention” is not meant to be limiting, but applies generally to any of the inventions defined in the claims or described herein. These terms are used interchangeably herein.
- glucan refers to a polysaccharide of D-glucose monomers that are linked by glycosidic linkages.
- glycosidic linkage and “glycosidic bond” are used
- alpha-1 ,3- glycosidic linkage refers to the type of covalent bond that joins alpha-D-glucose molecules to each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.
- alpha-1 , 6-glycosidic linkage refers to the type of covalent bond that joins alpha-D-glucose molecules to each other through carbons 1 and 6 on adjacent alpha-D-glucose rings.
- alpha-D-glucose will be referred to as "glucose.” All glycosidic linkages disclosed herein are alpha-glycosidic linkages, except where otherwise noted.
- the glycosidic linkage profile of a poly alpha-1 ,3-1 ,6-glucan herein can be determined using any method known in the art.
- a linkage profile can be determined using methods that use nuclear magnetic resonance (NMR) spectroscopy (e.g., 13 C NMR or 1 H NMR). These and other methods that can be used are disclosed in Food Carbohydrates: Chemistry. Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference.
- NMR nuclear magnetic resonance
- poly alpha-1 ,3-1 ,6-glucan alpha-1 , 3-1 ,6-glucan polymer
- poly (alpha-1 ,3)(alpha-1 , 6) glucan are used interchangeably herein (note that the order of the linkage denotations “1 ,3” and “1 ,6” in these terms is of no moment).
- Poly alpha-1 ,3-1 ,6-glucan herein is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 30% of the glycosidic linkages are alpha-1 ,3-glycosidic linkages, and at least about 30% of the glycosidic linkages are alpha-1 ,6-glycosidic linkages.
- Poly alpha-1 ,3-1 ,6-glucan is a type of polysaccharide containing a mixed glycosidic linkage content.
- poly alpha-1 ,3-1 ,6-glucan in certain embodiments herein excludes "alternan," which is a glucan containing alpha-1 ,3 linkages and alpha-1 ,6 linkages that consecutively alternate with each other (U.S. Pat. No. 5702942, U.S. Pat. Appl. Publ. No. 2006/0127328).
- Alpha-1 ,3 and alpha-1 ,6 linkages that "consecutively alternate" with each other can be visually represented by ...G-1 ,3-G-1 ,6-G-1 ,3-G-1 ,6-G-1 ,3-G-1 ,6-G-1 ,3-G-..., for example, where G represents glucose.
- Poly alpha-1 ,3-1 ,6-glucan herein can be produced by a glucosyltransferase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- a glucosyltransferase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- Such production can be from a gtf reaction herein.
- sucrose herein refers to a non-reducing disaccharide
- Sucrose is known commonly as table sugar.
- the "molecular weight" of a poly alpha-1 ,3-1 ,6-glucan or poly alpha-1 ,3- 1 ,6-glucan ether compound herein can be represented as number-average molecular weight (M n ) or as weight-average molecular weight (M w ).
- M n number-average molecular weight
- M w weight-average molecular weight
- molecular weight can be represented as Daltons, grams/mole, DP W (weight average degree of polymerization), or DP n (number average degree of
- HPLC high resolution chromatography
- SEC size exclusion chromatography
- GPC gel permeation chromatography
- gtf enzyme gtf enzyme catalyst
- gtf gtf
- glucansucrase The activity of a gtf enzyme herein catalyzes the reaction of the substrate sucrose to make the products poly alpha-1 ,3-1 ,6-glucan and fructose. Other products
- glucose where glucose is hydrolyzed from the glucosyl-gtf enzyme intermediate complex
- various soluble oligosaccharides include various soluble oligosaccharides, and leucrose (where glucose of the glucosyl-gtf enzyme intermediate complex is linked to fructose).
- Leucrose is a disaccharide composed of glucose and fructose linked by an alpha-1 ,5 linkage. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C- terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucan-binding domain.
- a gtf herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009).
- glucosyltransferase catalytic domain and “catalytic domain” are used interchangeably herein and refer to the domain of a glucosyltransferase enzyme that provides poly alpha-1 ,3-1 ,6-glucan-producing activity to the glucosyltransferase enzyme.
- a "gtf reaction solution” as used herein generally refers to a solution comprising at least one active glucosyltransferase enzyme in a solution comprising sucrose and water, and optionally other components. It is in a gtf reaction solution where the step of contacting water, sucrose and a glucosyltransferase enzyme is performed.
- the term "under suitable gtf reaction conditions” as used herein, refers to gtf reaction conditions that support conversion of sucrose to poly alpha-1 , 3-1 , 6-glucan via glucosyltransferase enzyme activity. A gtf reaction herein is not naturally occurring.
- percent by volume percent by volume
- volume percent percent by volume
- vol % percent by volume
- v/v % percent by volume
- Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.
- percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.
- increased refers to a greater quantity or activity such as a quantity or activity slightly greater than the original quantity or activity, or a quantity or activity in large excess compared to the original quantity or activity, and including all quantities or activities in between.
- these terms may refer to, for example, a quantity or activity that is at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is being compared.
- polynucleotide polynucleotide sequence
- nucleic acid sequence are used interchangeably herein. These terms encompass nucleotide sequences and the like.
- a polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
- a polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
- gene refers to a polynucleotide sequence that expresses a protein, and which may refer to the coding region alone or may include regulatory sequences upstream and/or downstream to the coding region (e.g., 5' untranslated regions upstream of the transcription start site of the coding region).
- a gene that is "native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; this gene is located in its natural location in the genome of an organism.
- Chimeric gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature.
- a “foreign” or “heterologous” gene refers to a gene that is introduced into the host organism by gene transfer.
- Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes.
- polynucleotide sequences in certain embodiments disclosed herein are heterologous.
- a "transgene” is a gene that has been introduced into the genome by a transformation procedure.
- a “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of particular host cell.
- recombinant or “heterologous” as used herein refers to an artificial combination of two otherwise separate segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
- recombinant “transgenic”, “transformed”, “engineered” or “modified for exogenous gene expression” are used interchangeably herein.
- transformation refers to the transfer of a nucleic acid molecule into a host organism.
- the nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of the host organism.
- Host organisms containing a transformed nucleic acid fragment(s) are "transgenic", “recombinant”, or “transformed”, and can be referred to as
- a native amino acid sequence or polynucleotide sequence is naturally occurring, whereas a non-native amino acid sequence or polynucleotide sequence does not occur in nature.
- Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
- Regulatory sequences refer to nucleotide sequences located upstream of the coding sequence's transcription start site, 5' untranslated regions and 3' non-coding regions, and which may influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5' untranslated leader sequence, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures and other elements involved in regulation of gene expression.
- sequence identity refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
- percentage of sequence identity refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
- BLAST Basic Local Alignment Search Tool
- NCBI National Center for Biotechnology Information
- BLASTN algorithm polynucleotide sequences
- BASTP algorithm polypeptide sequences
- percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV).
- ClustalW or ClustalV a Clustal algorithm
- EXTEND 0.5
- END GAP PENALTY false
- END GAP OPEN 10
- END GAP EXTEND 0.5 using a BLOSUM matrix (e.g., BLOSUM62).
- polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used.
- a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein.
- the variant amino acid sequence or polynucleotide sequence may have the same function/activity of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.
- isolated refers to any cellular component that has been completely or partially purified from its native source (e.g., an isolated polynucleotide or polypeptide molecule).
- an isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix.
- an isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a
- Another example is an isolated glucosyltransferase enzyme.
- poly alpha-1 ,3-1 ,6-glucan ether compound is a poly alpha-1 ,3-1 ,6-glucan that has been etherified with one or more organic groups such that the compound has a degree of substitution (DoS) with the organic group of about 0.05 to about 3.0.
- DoS degree of substitution
- Such etherification occurs at one or more hydroxyl groups of at least 30% of the glucose monomeric units of the poly alpha-1 ,3-1 ,6-glucan.
- a poly alpha-1 ,3-1 ,6-glucan ether compound is termed an "ether" herein by virtue of comprising the substructure -CG-O-C-, where "-CG-" represents a carbon atom of a glucose monomeric unit of a poly alpha-1 ,3-1 ,6-glucan ether compound (where such carbon atom was bonded to a hydroxyl group [-OH] in the poly alpha-1 ,3-1 ,6-glucan precursor of the ether), and where "-C-" is a carbon atom of the organic group.
- C G atoms 2, 4 and/or 6 of the glucose (G) may independently be linked to an OH group or be in ether linkage to an organic group.
- CG atoms 2, 4 and/or 6 of the glucose (G) may independently be linked to an OH group or be in ether linkage to an organic group.
- CG atoms 2, 3 and/or 4 of the glucose (G) may independently be linked to an OH group or be in ether linkage to an organic group.
- CG atoms 2, 3 and/or 4 of the glucose (G) may independently be linked to an OH group or be in ether linkage to an organic group.
- a "glucose" monomeric unit of a poly alpha- 1 ,3-1 ,6-glucan ether compound herein typically has one or more organic groups in ether linkage.
- a glucose monomeric unit can also be referred to as an etherized glucose monomeric unit.
- Poly alpha-1 ,3-1 ,6-glucan ether compounds disclosed herein are synthetic, man-made compounds.
- compositions comprising poly alpha-1 ,3-1 ,6-glucan e.g., isolated poly alpha-1 ,3-1 ,6-glucan
- an "organic group” group as used herein refers to a chain of one or more carbons that (i) has the formula -C N H 2n+ i (i.e., an alkyi group, which is completely saturated) or (ii) is mostly saturated but has one or more hydrogens substituted with another atom or functional group (i.e., a "substituted alkyi group”). Such substitution may be with one or more hydroxyl groups, oxygen atoms (thereby forming an aldehyde or ketone group), carboxyl groups, or other alkyi groups.
- an organic group herein can be an alkyi group, carboxy alkyi group, or hydroxy alkyi group.
- a “carboxy alkyi” group herein refers to a substituted alkyi group in which one or more hydrogen atoms of the alkyi group are substituted with a carboxyl group.
- a “hydroxy alkyi” group herein refers to a substituted alkyi group in which one or more hydrogen atoms of the alkyl group are substituted with a hydroxyl group.
- a "halide” herein refers to a compound comprising one or more halogen atoms (e.g., fluorine, chlorine, bromine, iodine).
- a halide herein can refer to a compound comprising one or more halide groups such as fluoride, chloride, bromide, or iodide.
- a halide group may serve as a reactive group of an etherification agent.
- reaction refers to a reaction comprising at least poly alpha-1 ,3-1 ,6-glucan and an etherification agent. These components are typically mixed (e.g., resulting in a slurry) and/or dissolved in a solvent (organic and/or aqueous) comprising alkali hydroxide. A reaction is placed under suitable conditions (e.g., time, temperature) for the etherification agent to etherify one or more hydroxyl groups of the glucose units of poly alpha-1 ,3-1 ,6-glucan with an organic group, thereby yielding a poly alpha-1 ,3-1 ,6-glucan ether compound.
- suitable conditions e.g., time, temperature
- alkaline conditions refers to a solution or mixture pH of at least 1 1 or 12. Alkaline conditions can be prepared by any means known in the art, such as by dissolving an alkali hydroxide in a solution or mixture.
- An etherification agent herein refers to an agent that can be used to etherify one or more hydroxyl groups of glucose units of poly alpha- 1 ,3-1 ,6-glucan with an organic group.
- An etherification agent thus comprises an organic group.
- poly alpha-1 ,3-1 ,6-glucan slurry refers to an aqueous mixture comprising the components of a glucosyltransferase enzymatic reaction such as poly alpha-1 ,3-1 ,6-glucan, sucrose, one or more glucosyltransferase enzymes, glucose and fructose.
- poly alpha-1 ,3-1 ,6-glucan wet cake herein refers to poly alpha- 1 ,3-1 ,6-glucan that has been separated from a slurry and washed with water or an aqueous solution. Poly alpha-1 ,3-1 ,6-glucan is not dried when preparing a wet cake.
- degree of substitution DoS as used herein refers to the average number of hydroxyl groups substituted in each monomeric unit (glucose) of a poly alpha-1 ,3-1 ,6-glucan ether compound.
- the degree of substitution in a poly alpha-1 ,3-1 ,6-glucan ether compound herein can be no higher than 3.
- M.S. molecular substitution
- M.S. can refer to the average moles of etherification agent used to react with each monomeric unit in poly alpha-1 ,3-1 ,6-glucan (M.S. can thus describe the degree of derivatization with an etherification agent). It is noted that the M.S. value for poly alpha-1 ,3-1 ,6-glucan may have no upper limit.
- hydroxyl group of the organic group may undergo further reaction, thereby coupling more of the organic group to the poly alpha-1 ,3-1 ,6-glucan.
- crosslink refers to a chemical bond, atom, or group of atoms that connects two adjacent atoms in one or more polymer molecules. It should be understood that, in a composition comprising crossl inked poly alpha- 1 ,3-1 ,6-glucan ether, crosslinks can be between at least two poly alpha-1 ,3-1 ,6- glucan ether molecules (i.e., intermolecular crosslinks); there can also be intramolecular crosslinking.
- a "crosslinking agent” as used herein is an atom or compound that can create crosslinks.
- hydrocolloid refers to a colloid system in which water is the dispersion medium.
- a hydrocolloid herein refers to a substance that is microscopically dispersed throughout another substance. Therefore, a hydrocolloid herein can also refer to a dispersion, mixture, or solution of poly alpha-1 ,3-1 ,6-glucan and/or one or more poly alpha-1 ,3-1 ,6-glucan ether compounds in water or aqueous solution.
- aqueous solution refers to a solution in which the solvent is water.
- Poly alpha-1 ,3-1 ,6-glucan and/or one or more poly alpha-1 ,3- 1 ,6-glucan ether compounds herein can be dispersed, mixed, and/or dissolved in an aqueous solution.
- An aqueous solution can serve as the dispersion medium of a hydrocolloid herein.
- viscosity refers to the measure of the extent to which a fluid or an aqueous composition such as a hydrocolloid resists a force tending to cause it to flow.
- Various units of viscosity that can be used herein include centipoise (cPs) and Pascal-second (Pa s).
- a centipoise is one one- hundredth of a poise; one poise is equal to 0.100 kg-m ⁇ 1 -s ⁇ 1 .
- viscosity modifier and “viscosity-modifying agent” as used herein refer to anything that can alter/modify the viscosity of a fluid or aqueous composition.
- shear thinning behavior refers to a decrease in the viscosity of the hydrocolloid or aqueous solution as shear rate increases.
- shear thickening behavior refers to an increase in the viscosity of the hydrocolloid or aqueous solution as shear rate increases.
- Shear rate herein refers to the rate at which a progressive shearing deformation is applied to the hydrocolloid or aqueous solution. A shearing deformation can be applied rotationally.
- contacting refers to any action that results in bringing together an aqueous composition and a poly alpha-1 , 3-1 ,6- glucan ether compound. Contacting can be performed by any means known in the art, such as dissolving, mixing, shaking, or homogenization, for example.
- Embodiments of the disclosed invention concern a reaction solution comprising water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1 , 3-1 , 6-glucan.
- the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- these enzymes can synthesize poly alpha-1 ,3-1 ,6-glucan that can be derivatized into ethers having enhanced viscosity modification qualities.
- poly alpha-1 ,3-1 ,6-glucan produced in a reaction solution herein:
- At least 30% of the glycosidic linkages of the poly alpha-1 , 3-1 , 6-glucan are alpha-1 ,6 linkages
- the poly alpha-1 , 3-1 , 6-glucan has a weight average degree of polymerization (DP W ) of at least 1000.
- At least 30% of the glycosidic linkages of poly alpha-1 ,3-1 ,6-glucan synthesized by a glucosyltransferase enzyme herein are alpha-1 ,3 linkages, and at least 30% of the glycosidic linkages are alpha-1 ,6 linkages.
- the percentage of alpha-1 , 3 linkages can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, or 64%.
- the percentage of alpha-1 ,6 linkages can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%.
- a poly alpha-1 ,3-1 ,6-glucan synthesized by a glucosyltransferase enzyme herein can have any one the aforementioned percentages of alpha-1 ,3 linkages and any one of the aforementioned percentages of alpha-1 ,6 linkages, just so long that the total of the percentages is not greater than 100%.
- the poly alpha-1 ,3-1 ,6-glucan can have (i) any one of 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (30%-40%) alpha-1 ,3 linkages and (ii) any one of 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% (60%-69%) alpha-1 ,6 linkages, just so long that the total of the percentages is not greater than 100%.
- Non-limiting examples include poly alpha-1 ,3-1 ,6-glucan with 31 % alpha-1 ,3 linkages and 67% alpha-1 ,6 linkages.
- alpha-1 ,3 and alpha-1 ,6 linkage profiles are provided in Table 2.
- at least 60% of the glycosidic linkages of poly alpha-1 ,3-1 ,6-glucan produced in a gtf reaction solution herein are alpha-1 ,6 linkages.
- Poly alpha-1 ,3-1 ,6-glucan synthesized by a glucosyltransferase enzyme herein can have, for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of glycosidic linkages other than alpha-1 ,3 and alpha-1 ,6.
- poly alpha-1 ,3-1 ,6-glucan only has alpha-1 ,3 and alpha-1 ,6 linkages.
- glucosyltransferase enzyme herein can be linear/unbranched.
- branches in the poly alpha-1 ,3-1 ,6-glucan can thus have no branch points or less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % branch points as a percent of the glycosidic linkages in the polymer.
- a glucosyltransferase enzyme can synthesize poly alpha-1 ,3-1 ,6-glucan comprising alpha-1 ,3 linkages and alpha-1 ,6 linkages that do not consecutively alternate with each other.
- G represents glucose
- glucosyltransferase enzyme herein can comprise, for example, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucose monomeric units that are linked consecutively with alternating alpha-1 ,3 and alpha-1 , 6 linkages.
- the molecular weight of poly alpha-1 ,3-1 ,6-glucan synthesized by a glucosyltransferase enzyme herein can be measured as DP W (weight average degree of polymerization) or DP n (number average degree of polymerization). Alternatively, molecular weight can be measured in Daltons or grams/mole. It may also be useful to refer to the number-average molecular weight (M n ) or weight-average molecular weight (M w ) of the poly alpha-1 ,3-1 ,6-glucan.
- Poly alpha-1 ,3-1 ,6-glucan synthesized by a glucosyltransferase enzyme herein has a DP W of at least about 1000.
- the DP W of the poly alpha- 1 ,3-1 ,6-glucan can be at least about 10000.
- the DP W can be at least about 1000 to about 15000.
- the DP W can be at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 1 1000, 12000, 13000, 14000, or 15000 (or any integer between 1000 and 15000), for example.
- poly alpha-1 ,3-1 ,6-glucan herein has a DP W of at least about 1000, such a glucan polymer is typically, but not necessarily, water- insoluble.
- 1 ,3-1 ,6-glucan can have an M w of at least about 50000, 100000, 200000,
- a glucosyltransferase enzyme herein may be obtained from any microbial source, such as a bacteria or fungus.
- bacterial glucosyltransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species.
- Streptococcus species include S.
- Leuconostoc species include L.
- Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L.
- curvatus L. plantarum, L. sakei, L brevis, L. buchneri, L. fermentum and L.
- a glucosyltransferase enzyme herein can comprise, or consist of, an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, wherein the
- glucosyltransferase enzyme has activity.
- a glucosyltransferase enzyme can comprise, or consist of, an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:28, or SEQ ID NO:30, wherein the
- glucosyltransferase enzyme has activity.
- a glucosyltransferase enzyme can comprise, or consist of, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- amino acids of the disclosed gtf enzyme sequences may be substituted with a conserved amino acid residue ("conservative amino acid substitution") as follows:
- glucosyltransferase enzymes for use in a gtf reaction solution may be any of the amino acid sequences disclosed herein and that further include 1 -300 (or any integer there between) residues on the N-terminus and/or C-terminus. Such additional residues may be from a corresponding wild type sequence from which the glucosyltransferase enzyme is derived, or may be another sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N-terminus), for example.
- amino acid sequence of a glucosyltransferase enzyme herein can be encoded by the polynucleotide sequence provided in SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, for example.
- amino acid sequence can be encoded by a polynucleotide sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.
- glucosyltransferase enzymes may be used to practice the disclosed invention.
- the glucosyltransferase enzyme does not have, or has very little (less than 1 %), alternansucrase activity, for example.
- a glucosyltransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyltransferase enzymes do not require the presence of a primer to perform glucan synthesis. A primer- dependent glucosyltransferase enzyme requires the presence of an initiating molecule in the reaction solution to act as a primer for the enzyme during glucan polymer synthesis.
- the term "primer” as used herein refers to any molecule that can act as the initiator for a glucosyltransferase enzyme. Oligosaccharides and polysaccharides can serve a primers, for example.
- Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan, for example.
- Dextran for use as a primer can be dextran T10 (i.e., dextran having a molecular weight of 10 kD), for example.
- dextran primer can have a molecular weight of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 25 kD, for example.
- a glucosyltransferase enzyme of the disclosed invention may be produced by any means known in the art.
- the glucosyltransferase enzyme may be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system.
- heterologous expression system such as a microbial heterologous expression system.
- expression systems include bacterial (e.g., E. coli such as TOP10 or MG1655; Bacillus sp.) and eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) expression systems.
- E. coli such as TOP10 or MG1655
- Bacillus sp. Bacillus sp.
- eukaryotic e.g., yeasts such as Pichia sp. and Saccharomyces sp.
- a glucosyltransferase enzyme described herein may be used in any purification state (e.g., pure or non-pure).
- the glucosyltransferase enzyme may be purified and/or isolated prior to its use.
- Examples of glucosyltransferase enzymes that are non-pure include those in the form of a cell lysate.
- a cell lysate or extract may be prepared from a bacteria (e.g., E. coli) used to heterologously express the enzyme.
- the bacteria may be subjected to disruption using a French pressure cell.
- bacteria may be homogenized with a homogenizer (e.g., APV, Rannie, Gaulin).
- a glucosyltransferase enzyme is typically soluble in these types of preparations.
- a bacterial cell lysate, extract, or homogenate herein may be used at about 0.15- 0.3% (v/v) in a reaction solution for producing poly alpha-1 ,3-1 ,6-glucan from sucrose.
- a heterologous gene expression system in certain embodiments may be one that is designed for protein secretion.
- the glucosyltransferase enzyme comprises a signal peptide (signal sequence) in such embodiments.
- the signal peptide may be either its native signal peptide or a heterologous signal peptide.
- glucosyltransferase enzyme activity can be determined using any method known in the art.
- glucosyltransferase enzyme activity can be determined by measuring the production of reducing sugars (fructose and glucose) in a reaction solution containing sucrose (-50 g/L), dextran T10 ( ⁇ 1 mg/mL) and potassium phosphate buffer ( ⁇ pH 6.5, 50 mM), where the solution is held at -22-25 °C for -24-30 hours.
- the reducing sugars can be measured by adding 0.01 ml_ of the reaction solution to a mixture containing -1 N NaOH and -0.1 % triphenyltetrazolium chloride and then monitoring the increase in absorbance at OD 48 onm for -five minutes.
- the temperature of a gtf reaction solution herein can be controlled, if desired. In certain embodiments, the temperature is between about 5 °C to about 50 °C. The temperature in certain other embodiments is between about 20 °C to about 40 °C. Alternatively, the temperature may be about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 °C.
- the temperature of a gtf reaction solution herein may be maintained using various means known in the art.
- the temperature can be
- the initial concentration of sucrose in a gtf reaction solution herein can be about 20 g/L to about 400 g/L, for example.
- the initial concentration of sucrose can be about 75 g/L to about 175 g/L, or from about 50 g/L to about 150 g/L.
- the initial concentration of sucrose can be about 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, or 160 g/L (or any integer between 40 and 160 g/L), for example.
- “Initial concentration of sucrose” refers to the sucrose concentration in a gtf reaction solution just after all the reaction solution components have been added (water, sucrose, gtf enzyme).
- sucrose can be highly pure (> 99.5%), have a purity of at least 99.0%, or be reagent grade sucrose.
- Sucrose for use herein may be derived from any renewable sugar source such as sugar cane, sugar beets, cassava, sweet sorghum, or corn.
- the sucrose can be provided in any form such as crystalline form or non-crystalline form (e.g., syrup or cane juice).
- the pH of a gtf reaction solution in certain embodiments can be between about 4.0 to about 8.0.
- the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
- the pH can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: phosphate, tris, citrate, or a combination thereof.
- Buffer concentration in a gtf reaction solution can be from 0 mM to about 100 mM, or about 10, 20, or 50 mM, for example.
- the disclosed invention also concerns a method for producing poly alpha- 1 ,3-1 ,6-glucan comprising the step of contacting at least water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1 ,3-1 ,6-glucan.
- the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- Poly alpha-1 , 3-1 , 6-glucan is produced in the contacting step. This poly alpha-1 ,3-1 ,6-glucan can optionally be isolated.
- the contacting step in a method herein of producing poly alpha-1 ,3-1 ,6- glucan can comprise providing a gtf reaction solution comprising water, sucrose and any glucosyltransferase enzyme disclosed herein. It would be understood that, as the glucosyltransferase enzyme synthesizes poly alpha-1 ,3-1 ,6-glucan, the reaction solution typically becomes a reaction mixture given that insoluble poly alpha-1 ,3-1 ,6-glucan falls out of solution as indicated by clouding of the reaction.
- the contacting step of the disclosed method can be performed in any number of ways. For example, the desired amount of sucrose can first be dissolved in water (optionally, other components may also be added at this stage of preparation, such as buffer components), followed by addition of the
- the solution may be kept still, or agitated via stirring or orbital shaking, for example.
- the reaction can be, and typically is, cell- free.
- Completion of a gtf reaction in certain embodiments can be determined visually (e.g., no more accumulation of precipitated poly alpha-1 ,3-1 ,6-glucan) and/or by measuring the amount of sucrose left in the solution (residual sucrose), where a percent sucrose consumption of over about 90% can indicate reaction completion.
- a reaction of the disclosed process can take about 12, 18, 24, 30, 36, 48, 60, 72, 84, or 96 hours to complete. Reaction time may depend, for example, on certain parameters such as the amount of sucrose and
- the yield of poly alpha-1 ,3-1 ,6-glucan produced in a gtf reaction in certain embodiments herein can be at least about 4%, 5%, 6%, 7%, or 8%, based on the weight of the sucrose used in the reaction solution.
- Poly alpha-1 ,3-1 ,6-glucan produced in the disclosed method may optionally be isolated.
- insoluble poly alpha-1 ,3-1 ,6-glucan may be separated by centrifugation or filtration. In doing so, the poly alpha-1 ,3-1 ,6- glucan is separated from the rest of the reaction solution, which may comprise water, fructose and certain byproducts (e.g., leucrose, soluble oligosaccharides). This solution may also comprise glucose monomer and residual sucrose.
- the linkage profile and/or molecular weight of poly alpha-1 ,3-1 ,6-glucan produced in a gtf reaction herein can be any of those disclosed above.
- at least 30% of the glycosidic linkages are alpha-1 ,3 linkages
- at least 30% of the glycosidic linkages are alpha-1 ,6 linkages
- the poly alpha-1 ,3-1 ,6-glucan has a DP W of at least 1000.
- Poly alpha-1 ,3-1 ,6-glucan produced in a gtf reaction can have at least 60% alpha-1 ,6 linkages, and/or have a DPw of at least about 10000.
- the poly alpha-1 , 3-1 , 6-glucan has a weight average degree of polymerization (DP W ) of at least 1000;
- poly alpha-1 ,3-1 ,6-glucan disclosed herein can be
- At least 30% of the glycosidic linkages of poly alpha-1 ,3-1 ,6-glucan disclosed herein are alpha-1 , 3 linkages, and at least 30% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan are alpha-1 ,6 linkages.
- the percentage of alpha-1 ,3 linkages in poly alpha-1 ,3-1 ,6-glucan herein can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, or 64%.
- the percentage of alpha-1 ,3 linkages in poly alpha-1 ,3-1 ,6-glucan herein can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 5
- percentage of alpha-1 ,6 linkages in poly alpha-1 ,3-1 ,6-glucan herein can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%.
- a poly alpha-1 ,3-1 ,6-glucan of the invention can have any one the aforementioned percentages of alpha-1 ,3 linkages and any one of the
- poly alpha-1 ,3-1 ,6- glucan herein can have (i) any one of 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (30%-40%) alpha-1 ,3 linkages and (ii) any one of 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% (60%-69%) alpha-1 ,6 linkages, just so long that the total of the percentages is not greater than 100%.
- Non-limiting examples include poly alpha-1 ,3-1 ,6-glucan with 31 % alpha-1 ,3 linkages and 67% alpha-1 ,6 linkages.
- Other examples of alpha-1 ,3 and alpha- 1 ,6 linkage profiles are provided in Table 2.
- at least 60% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan are alpha-1 ,6 linkages.
- a poly alpha-1 ,3-1 ,6-glucan of the invention can have, for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of glycosidic linkages other than alpha-1 ,3 and alpha-1 ,6.
- a poly alpha-1 ,3-1 ,6- glucan only has alpha-1 , 3 and alpha-1 ,6 linkages.
- the backbone of a poly alpha-1 ,3-1 ,6-glucan disclosed herein can be linear/unbranched. Alternatively, there can be branches in the poly alpha-1 , 3- 1 ,6-glucan.
- a poly alpha-1 ,3-1 ,6-glucan in certain embodiments can thus have no branch points or less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % branch points as a percent of the glycosidic linkages in the polymer.
- alpha-1 ,3 linkages and alpha-1 ,6 linkages of a poly alpha-1 ,3-1 ,6- glucan in the disclosed composition do not consecutively alternate with each other.
- G represents glucose
- Poly alpha-1 , 3-1 ,6-glucan in certain embodiments herein comprises less than 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucose monomeric units that are linked consecutively with alternating alpha-1 ,3 and alpha-1 ,6 linkages.
- the molecular weight of a poly alpha-1 ,3-1 ,6-glucan disclosed herein can be measured as DP W (weight average degree of polymerization) or DP n (number average degree of polymerization). Alternatively, molecular weight can be measured in Daltons or grams/mole. It may also be useful to refer to the number-average molecular weight (M n ) or weight-average molecular weight (M w ) of the poly alpha-1 ,3-1 ,6-glucan.
- a poly alpha-1 ,3-1 ,6-glucan herein has a DP W of at least about 1000.
- the DP W of the poly alpha-1 ,3-1 ,6-glucan can be at least about 10000.
- the DP W can be at least about 1000 to about 15000. Alternatively still, the DP W can be at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000,
- a poly alpha-1 ,3-1 ,6-glucan herein has a DP W of at least about 1000, such a glucan polymer is typically, but not necessarily, water-insoluble.
- a poly alpha-1 ,3-1 ,6-glucan herein can have an M w of at least about
- the M w in certain embodiments is at least about 1000000.
- a poly alpha-1 ,3-1 ,6-glucan herein can comprise at least 6 glucose monomeric units, for example.
- the number of glucose monomeric units can be at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000,
- Poly alpha-1 ,3-1 ,6-glucan herein can be produced, for example, using a glucosyltransferase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the glucosyltransferase enzyme can comprise an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- Production of poly alpha-1 ,3-1 ,6- glucan of the disclosed invention can be accomplished with a gtf reaction as disclosed herein, for example.
- Poly alpha-1 ,3-1 ,6-glucan herein can be provided in the form of a powder when dry, or a paste, colloid or other dispersion when wet, for example.
- a composition comprising poly alpha-1 ,3-1 ,6-glucan in certain embodiments is one in which the constituent poly alpha-1 ,3-1 ,6-glucan behaves as a thickening agent. It is believed that poly alpha-1 , 3-1 , 6-glucan herein is suitable as a thickening agent, which is a substance that absorbs liquids such as water and swells upon such absorption. Swelling of poly alpha-1 ,3-1 ,6-glucan in a liquid can yield a slurry or colloid, for example.
- a composition comprising poly alpha-1 ,3-1 ,6-glucan may be in the form of a personal care product, pharmaceutical product, food product, household product, or industrial product, such as any of those products disclosed below for the application of ether derivatives of poly alpha-1 ,3-1 ,6-glucan.
- the amount of poly alpha-1 ,3-1 ,6-glucan in the composition can be, for example, about 0.1 -10 wt%, 0.1 -5 wt%, 0.1 -4 wt%, 0.1 -3 wt%, 0.1 -2 wt%, or 0.1 -1 wt%, or an amount that provides the desired degree of thickening to the composition.
- the poly alpha-1 ,3-1 ,6-glucan ether compound has a weight average degree of polymerization (DP W ) of at least 1000; (iv) the alpha-1 ,3 linkages and alpha-1 ,6 linkages of the poly alpha-1 ,3-
- the poly alpha-1 ,3-1 ,6-glucan ether compound has a degree of substitution (DoS) with an organic group of about 0.05 to about 3.0.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein has enhanced viscosity modification qualities such as the ability to viscosify an aqueous composition at low concentration.
- a poly alpha-1 ,3- 1 ,6-glucan ether compound herein can have a relatively low DoS and still be an effective viscosity modifier.
- At least 30% of the glycosidic linkages of a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein are alpha-1 ,3 linkages, and at least 30% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan ether compound are alpha- 1 ,6 linkages.
- the percentage of alpha-1 ,3 linkages in a poly alpha- 1 ,3-1 ,6-glucan ether compound herein can be at least 31 %, 32%, 33%, 34%,
- the percentage of alpha-1 ,6 linkages in a poly alpha-1 ,3-1 ,6-glucan ether compound herein can be at least 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%.
- a poly alpha-1 ,3-1 ,6-glucan ether compound of the invention can have any one the aforementioned percentages of alpha-1 ,3 linkages and any one of the aforementioned percentages of alpha-1 ,6 linkages, just so long that the total of the percentages is not greater than 100%.
- the poly alpha-1 ,3- 1 ,6-glucan ether compound can have (i) any one of 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (30%-40%) alpha-1 ,3 linkages and (ii) any one of 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% (60%-69%) alpha-1 ,6 linkages, just so long that the total of the percentages is not greater than 100%.
- Non-limiting examples include poly alpha-1 , 3-1 , 6-glucan ether compounds with 31 % alpha-1 ,3 linkages and 67% alpha-1 ,6 linkages.
- alpha-1 ,3 and alpha-1 ,6 linkage profiles of certain poly alpha-1 ,3- 1 ,6-glucan ether compounds herein are provided in Table 2, which discloses linkage profiles of isolated poly alpha-1 ,3-1 ,6-glucan that can be used to prepare the disclosed ethers.
- at least 60% of the glycosidic linkages of the poly alpha-1 ,3-1 ,6-glucan ether compound are alpha-1 ,6 linkages.
- a poly alpha-1 ,3-1 ,6-glucan ether compound of the invention can have, for example, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of glycosidic linkages other than alpha-1 ,3 and alpha-1 ,6.
- a poly alpha-1 ,3-1 ,6-glucan ether compound only has alpha-1 ,3 and alpha-1 ,6 linkages.
- the backbone of a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein can be linear/unbranched. Alternatively, there can be branches in the poly alpha-1 ,3-1 ,6-glucan ether compound.
- a poly alpha-1 ,3-1 ,6-glucan ether compound in certain embodiments can thus have no branch points or less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % branch points as a percent of the glycosidic linkages in the polymer.
- alpha-1 ,3 linkages and alpha-1 ,6 linkages of a poly alpha-1 ,3-1 ,6- glucan ether compound disclosed herein do not consecutively alternate with each other.
- G represents etherized glucose
- Poly alpha-1 ,3-1 ,6-glucan ether compounds in certain embodiments herein less than 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glucose
- the molecular weight of a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein can be measured as DP W (weight average degree of
- molecular weight can be measured in Daltons or grams/mole. It may also be useful to refer to the number-average molecular weight (M n ) or weight-average molecular weight (M w ) of the poly alpha-1 ,3-1 ,6-glucan ether compound.
- a poly alpha-1 ,3-1 ,6-glucan ether compound herein has a DP W of at least about 1000.
- the DP W of the poly alpha-1 ,3-1 ,6-glucan ether compound can be at least about 10000.
- the DP W can be at least about 1000 to about 15000.
- the DP W can be at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 1 1000, 12000, 13000, 14000, or 15000 (or any integer between 1000 and 15000), for example.
- a poly alpha-1 ,3-1 ,6-glucan ether compound herein can have an M w of at least about 50000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 1 100000, 1200000, 1300000, 1400000, 1500000, or 1600000 (or any integer between 50000 and 1600000), for example.
- the M w in certain embodiments is at least about 1000000.
- a poly alpha-1 ,3-1 ,6-glucan ether compound herein can comprise at least 6 glucose monomeric units (most of such units typically contain ether-linked organic groups), for example.
- the number of glucose monomeric units can be at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 (or any integer between 10 and 9000), for example.
- Poly alpha-1 ,3-1 ,6-glucan ether compounds of the invention have a DoS with an organic group of about 0.05 to about 3.0.
- the DoS of a poly alpha-1 ,3-1 ,6-glucan ether compound can be about 0.3 to 1 .0.
- the DoS can alternatively be at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.
- the percentage of glucose monomeric units of a poly alpha-1 ,3-1 ,6-glucan ether compound herein that are ether-linked to an organic group can vary depending on the degree to which a poly alpha-1 , 3- 1 ,6-glucan is etherified with an organic group in an etherification reaction. This percentage can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (or any integer value between 30% and 100%), for example.
- glucose monomeric unit of an ether compound may independently be linked to an OH group or be in ether linkage to an organic group.
- An organic group herein may be an alkyl group such as a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl group, for example.
- an organic group may be a substituted alkyl group in which there is a substitution on one or more carbons of the alkyl group.
- substitution(s) may be one or more hydroxyl, aldehyde, ketone, and/or carboxyl groups.
- a substituted alkyl group may be a hydroxy alkyl group, dihydroxy alkyl group, or carboxy alkyl group.
- Suitable hydroxy alkyl groups are hydroxymethyl (-CH 2 OH), hydroxyethyl (e.g., -CH 2 CH 2 OH, -CH(OH)CH 3 ), hydroxypropyl (e.g.,
- dihydroxy alkyl groups such as dihydroxymethyl, dihydroxyethyl (e.g., -CH(OH)CH 2 OH), dihydroxypropyl (e.g., -CH 2 CH(OH)CH 2 OH, -CH(OH)CH(OH)CH 3 ), dihydroxybutyl and
- Examples of suitable carboxy alkyl groups are carboxymethyl
- carboxypropyl e.g., -CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 3 ,
- one or more carbons of an alkyl group can have a substitution(s) with another alkyl group.
- substituent alkyl groups are methyl, ethyl and propyl groups.
- an organic group can be -CH(CH 3 )CH 2 CH 3 or -CH 2 CH(CH 3 )CH 3 , for example, which are both propyl groups having a methyl substitution.
- a substitution (e.g., hydroxy or carboxy group) on an alkyl group in certain embodiments may be bonded to the terminal carbon atom of the alkyl group, where the terminal carbon group is opposite the terminus that is in ether linkage to a glucose monomeric unit in a poly alpha-1 ,3-1 ,6-glucan ether compound.
- An example of this terminal substitution is the hydroxypropyl group -CH2CH2CH2OH.
- a substitution may be on an internal carbon atom of an alkyl group.
- An example on an internal substitution is the hydroxypropyl group -CH 2 CH(OH)CH 3 .
- An alkyl group can have one or more substitutions, which may be the same (e.g., two hydroxyl groups [dihydroxy]) or different (e.g., a hydroxyl group and a carboxyl group).
- Poly alpha-1 ,3-1 ,6-glucan ether compounds in certain embodiments disclosed herein may contain one type of organic group.
- Examples of such compounds contain a carboxy alkyl group as the organic group (carboxyalkyi poly alpha-1 ,3-1 ,6-glucan, generically speaking).
- a specific non-limiting example of such a compound is carboxymethyl poly alpha-1 , 3-1 ,6-glucan.
- poly alpha-1 , 3-1 ,6-glucan ether compounds disclosed herein can contain two or more different types of organic groups.
- examples of such compounds contain (i) two different alkyl groups as organic groups, (ii) an alkyl group and a hydroxy alkyl group as organic groups (alkyl hydroxyalkyi poly alpha-1 ,3-1 ,6-glucan, generically speaking), (iii) an alkyl group and a carboxy alkyl group as organic groups (alkyl carboxyalkyi poly alpha-1 ,3-1 ,6-glucan, generically speaking), (iv) a hydroxy alkyl group and a carboxy alkyl group as organic groups (hydroxyalkyi carboxyalkyi poly alpha-1 ,3-1 ,6-glucan, generically speaking), (v) two different hydroxy alkyl groups as organic groups, or (vi) two different carboxy alkyl groups as organic groups.
- poly alpha-1 ,3-1 ,6-glucan ether compounds may be derived from any poly alpha-1 ,3-1 ,6-glucan disclosed herein.
- a poly alpha-1 ,3-1 ,6-glucan ether compound of the invention can be produced by ether-derivatizing poly alpha-1 ,3-1 ,6-glucan using an etherification reaction as disclosed herein.
- the poly alpha-1 ,3-1 ,6- glucan from which a poly alpha-1 ,3-1 ,6-glucan ether compound is derived is a product of a glucosyltransferase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the glucosyltransferase enzyme can comprise an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- a composition comprising a poly alpha-1 ,3-1 ,6-glucan ether compound can be a hydrocolloid or aqueous solution having a viscosity of at least about 10 cPs.
- a hydrocolloid or aqueous solution has a viscosity of at least about 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3500, or 4000 cPs (or any integer between 100 and 4000 cPs), for example.
- Viscosity can be measured with the hydrocolloid or aqueous solution at any temperature between about 3 °C to about 1 10 °C (or any integer between 3 and 1 10 °C). Alternatively, viscosity can be measured at a temperature between about 4 °C to 30 °C, or about 20 °C to 25 °C. Viscosity can be measured at atmospheric pressure (about 760 torr) or any other higher or lower pressure.
- the viscosity of a hydrocolloid or aqueous solution disclosed herein can be measured using a viscometer or rheometer, or using any other means known in the art. It would be understood by those skilled in the art that a viscometer or rheometer can be used to measure the viscosity of those hydrocolloids and aqueous solutions of the invention that exhibit shear thinning behavior or shear thickening behavior (i.e., liquids with viscosities that vary with flow conditions).
- the viscosity of such embodiments can be measured at a rotational shear rate of about 10 to 1000 rpm (revolutions per minute) (or any integer between 10 and 1000 rpm), for example. Alternatively, viscosity can be measured at a rotational shear rate of about 10, 60, 150, 250, or 600 rpm.
- pH of a hydrocolloid or aqueous solution disclosed herein can be between about 2.0 to about 12.0.
- pH can be about 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 1 1 .0, 12.0; between about 4.0 and 8.0; or between about 5.0 and 8.0.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein can be present in a hydrocolloid or aqueous solution at a weight percentage (wt%) of at least about 0.01 %, 0.05%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 .0%, 1 .2%, 1 .4%, 1 .6%, 1 .8%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, for example.
- a hydrocolloid or aqueous solution herein can comprise other components in addition to a poly alpha-1 ,3-1 ,6-glucan ether compound.
- the hydrocolloid or aqueous solution can comprise one or more salts such as a sodium salt (e.g., NaCI, Na 2 SO ).
- salts include those having (i) an aluminum, ammonium, barium, calcium, chromium (II or III), copper (I or II), iron (II or III), hydrogen, lead (II), lithium, magnesium, manganese (II or III), mercury (I or II), potassium, silver, sodium strontium, tin (II or IV), or zinc cation, and (ii) an acetate, borate, bromate, bromide, carbonate, chlorate, chloride, chlorite, chromate, cyanamide, cyanide, dichromate, dihydrogen phosphate, ferricyanide, ferrocyanide, fluoride, hydrogen carbonate, hydrogen phosphate, hydrogen sulfate, hydrogen sulfide, hydrogen sulfite, hydride, hydroxide, hypochlorite, iodate, iodide, nitrate, nitride, nitrite, oxalate, oxide, perchlorate, perman
- any salt having a cation from (i) above and an anion from (ii) above can be in a hydrocolloid or aqueous solution, for example.
- a salt can be present in a hydrocolloid or aqueous solution at a wt% of about .01 % to about 10.00% (or any hundredth increment between .01 and 10.00).
- a poly alpha-1 , 3-1 , 6-glucan ether compound can be in an anionic form in the hydrocolloid or aqueous solution. Examples may include those poly alpha-1 ,3-1 , 6-glucan ether compounds having an organic group comprising an alkyl group substituted with a carboxyl group. Carboxyl (COOH) groups in a carboxyalkyl poly alpha-1 ,3-1 ,6-glucan ether compound can convert to
- a poly alpha-1 ,3-1 ,6-glucan ether compound can be a sodium carboxyalkyl poly alpha-1 ,3-1 , 6-glucan ether (e.g., sodium carboxymethyl poly alpha-1 ,3-1 ,6-glucan), potassium carboxyalkyl poly alpha- 1 ,3-1 ,6-glucan ether (e.g., potassium carboxymethyl poly alpha-1 ,3-1 ,6-glucan), or lithium carboxyalkyl poly alpha-1 ,3-1 ,6-glucan ether (e.g., lithium
- carboxymethyl poly alpha-1 ,3-1 ,6-glucan for example.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein may be crosslinked using any means known in the art.
- Such crosslinks may be borate crosslinks, where the borate is from any boron-containing compound (e.g., boric acid, diborates, tetraborates, pentaborates, polymeric compounds such as Polybor ® , polymeric compounds of boric acid, alkali borates).
- crosslinks can be provided with polyvalent metals such as titanium or zirconium. Titanium crosslinks may be provided using titanium IV-containing compounds such as titanium ammonium lactate, titanium triethanolamine, titanium
- Zirconium crosslinks can be provided using zirconium IV-containing compounds such as zirconium lactate, zirconium carbonate, zirconium acetylacetonate, zirconium
- crosslinks can be provided with any crosslinking agent described in U.S. Patent Nos. 4,462,917; 4,464,270; 4,477,360 and 4,799,550; which are all incorporated herein by reference.
- a crosslinking agent e.g., borate
- borate may be present in a hydrocolloid or aqueous solution at a concentration of about 0.2% to 20 wt%, or about 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 wt%, for example.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein that is crosslinked typically has a higher viscosity in an aqueous solution compared to its non-crossl inked counterpart.
- a crosslinked poly alpha-1 ,3-1 ,6-glucan ether compound can have increased shear thickening behavior compared to its non-crossl inked counterpart.
- Hydrocolloids and aqueous solutions in certain embodiments of the invention are believed to have either shear thinning behavior or shear thickening behavior.
- Shear thinning behavior is observed as a decrease in viscosity of the hydrocolloid or aqueous solution as shear rate increases, whereas shear thickening behavior is observed as an increase in viscosity of the hydrocolloid or aqueous solution as shear rate increases.
- Modification of the shear thinning behavior or shear thickening behavior of an aqueous solution herein is due to the admixture of a poly alpha-1 ,3-1 ,6-glucan ether to the aqueous composition.
- one or more poly alpha-1 ,3-1 ,6-glucan ether compounds of the invention can be added to an aqueous liquid, solution, or mixture to modify its rheological profile (i.e., the flow properties of the aqueous liquid, solution, or mixture are modified). Also, one or more poly alpha-1 ,3-1 ,6-glucan ether compounds of the invention can be added to an aqueous liquid, solution, or mixture to modify its viscosity.
- the rheological properties of hydrocolloids and aqueous solutions of the invention can be observed by measuring viscosity over an increasing rotational shear rate (e.g., from about 10 rpm to about 250 rpm).
- shear thinning behavior of a hydrocolloid or aqueous solution disclosed herein can be observed as a decrease in viscosity (cPs) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (or any integer between 5% and 95%) as the rotational shear rate increases from about 10 rpm to 60 rpm, 10 rpm to 150 rpm, 10 rpm to 250 rpm, 60 rpm to 150 rpm, 60 rpm to 250 rpm, or 150 rpm to 250 rpm.
- shear thickening behavior of a hydrocolloid or aqueous solution disclosed herein can be observed as an increase in viscosity (cPs) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% (or any integer between 5% and 200%) as the rotational shear rate increases from about 10 rpm to 60 rpm, 10 rpm to 150 rpm, 10 rpm to 250 rpm, 60 rpm to 150 rpm, 60 rpm to 250 rpm, or 150 rpm to 250 rpm.
- cPs viscosity
- a hydrocolloid or aqueous solution disclosed herein can be in the form of, and/or comprised in, a personal care product, pharmaceutical product, food product, household product, or industrial product.
- Poly alpha-1 ,3-1 ,6-glucan ether compounds disclosed herein can be used as thickening agents in each of these products.
- Such a thickening agent may be used in conjunction with one or more other types of thickening agents if desired, such as those disclosed in U.S. Patent No. 8541041 , the disclosure of which is incorporated herein by reference in its entirety.
- Personal care products herein are not particularly limited and include, for example, skin care compositions, cosmetic compositions, antifungal, and/or
- Personal care products herein may be in the form of, for example, lotions, creams, pastes, balms, ointments, pomades, gels, liquids, combinations of these and the like.
- the personal care products disclosed herein can include at least one active ingredient.
- An active ingredient is generally recognized as an ingredient that causes the intended pharmacological effect.
- a skin care product can be applied to skin for addressing skin damage related to a lack of moisture.
- a skin care product may also be used to address the visual appearance of skin (e.g., reduce the appearance of flaky, cracked, and/or red skin) and/or the tactile feel of the skin (e.g., reduce roughness and/or dryness of the skin while improved the softness and subtleness of the skin).
- a skin care product typically may include at least one active ingredient for the treatment or prevention of skin ailments, providing a cosmetic effect, or for providing a moisturizing benefit to skin, such as zinc oxide, petrolatum, white petrolatum, mineral oil, cod liver oil, lanolin, dimethicone, hard fat, vitamin A, allantoin, calamine, kaolin, glycerin, or colloidal oatmeal, and combinations of these.
- a skin care product may include one or more natural moisturizing factors such as ceramides, hyaluronic acid, glycerin, squalane, amino acids, cholesterol, fatty acids, triglycerides, phospholipids,
- glycosphingolipids urea, linoleic acid, glycosaminoglycans, mucopolysaccharide, sodium lactate, or sodium pyrrolidone carboxylate, for example.
- Other molecules glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharide, sodium lactate, or sodium pyrrolidone carboxylate, for example.
- ingredients that may be included in a skin care product include, without limitation, glycerides, apricot kernel oil, canola oil, squalane, squalene, coconut oil, corn oil, jojoba oil, jojoba wax, lecithin, olive oil, safflower oil, sesame oil, shea butter, soybean oil, sweet almond oil, sunflower oil, tea tree oil, shea butter, palm oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil.
- glycerides apricot kernel oil, canola oil, squalane, squalene, coconut oil, corn oil, jojoba oil, jojoba wax, lecithin, olive oil, safflower oil, sesame oil, shea butter, soybean oil, sweet almond oil, sunflower oil, tea tree oil, shea butter, palm oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil.
- a personal care product herein can also be in the form of makeup or other product including, but not limited to, a lipstick, mascara, rouge, foundation, blush, eyeliner, lip liner, lip gloss, other cosmetics, sunscreen, sun block, nail polish, mousse, hair spray, styling gel, nail conditioner, bath gel, shower gel, body wash, face wash, shampoo, hair conditioner (leave-in or rinse-out), cream rinse, hair dye, hair coloring product, hair shine product, hair serum, hair anti-frizz product, hair split-end repair product, lip balm, skin conditioner, cold cream, moisturizer, body spray, soap, body scrub, exfoliant, astringent, scruffing lotion, depilatory, permanent waving solution, antidandruff formulation, antiperspirant composition, deodorant, shaving product, pre-shaving product, after-shaving product, cleanser, skin gel, rinse, toothpaste, or mouthwash, for example.
- a lipstick mascara, rouge, foundation, blush, eyeliner, lip liner, lip gloss
- other cosmetics sunscreen
- a pharmaceutical product herein can be in the form of an emulsion, liquid, elixir, gel, suspension, solution, cream, or ointment, for example. Also, a pharmaceutical product herein can be in the form of any of the personal care products disclosed herein. A pharmaceutical product can further comprise one or more pharmaceutically acceptable carriers, diluents, and/or pharmaceutically acceptable salts.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein can also be used in capsules, encapsulants, tablet coatings, and as an excipients for medicaments and drugs.
- Non-limiting examples of food products herein include vegetable, meat, and soy patties; reformed seafood; reformed cheese sticks; cream soups;
- batters for fried foods, pancakes/waffles and cakes pet foods; beverages; frozen desserts; ice cream; cultured dairy products such as cottage cheese, yogurt, cheeses, and sour creams; cake icing and glazes; whipped topping; leavened and unleavened baked goods; and the like.
- Poly alpha-1 ,3-1 ,6-glucan ether compounds, hydrocolloids and aqueous compositions disclosed herein can be used to provide one or more of the following physical properties to a food product (or any personal care product, pharmaceutical product, or industrial product): thickening, freeze/thaw stability, lubricity, moisture retention and release, film formation, texture, consistency, shape retention, emulsification, binding, suspension, and gelation, for example.
- Poly alpha-1 ,3-1 ,6-glucan ether compounds disclosed herein can typically be used in a food product at a level of about 0.01 to about 5 wt%, for example.
- a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein can be comprised in a foodstuff or any other ingestible material (e.g., enteral
- the concentration or amount of a poly alpha-1 ,3-1 ,6- glucan ether compound in a product, on a weight basis can be about 0.1 -3 wt%, 0.1 -4 wt%, 0.1 -5 wt%, or 0.1 -10 wt%.
- a household and/or industrial product herein can be in the form of drywall tape-joint compounds; mortars; grouts; cement plasters; spray plasters; cement stucco; adhesives; pastes; wall/ceiling texturizers; binders and processing aids for tape casting, extrusion forming, and injection molding and ceramics; spray adherents and suspending/dispersing aids for pesticides, herbicides, and fertilizers; fabric softeners; laundry detergents; hard surface cleaners; air fresheners; polymer emulsions; gels such as water-based gels; surfactant solutions; paints such as water-based paints; protective coatings; adhesives; sealants and caulks; inks such as water-based ink; metal-working fluids;
- emulsion-based metal cleaning fluids used in electroplating, phosphatizing, galvanizing and/or general metal cleaning operations; hydraulic fluids (e.g., those used for fracking in downhole operations); and aqueous mineral slurries, for example.
- the disclosed invention also concerns a method for increasing the viscosity of an aqueous composition.
- This method comprises contacting one or more poly alpha-1 ,3-1 ,6-glucan ether compounds with the aqueous composition, wherein:
- the poly alpha-1 ,3-1 ,6-glucan ether compound has a weight average degree of polymerization (DP W ) of at least 1000;
- the contacting step in this method results in increasing the viscosity of the aqueous composition.
- Any hydrocolloid and aqueous solution disclosed herein can be produced using this method.
- An aqueous composition herein can be water (e.g., de-ionized water), an aqueous solution, or a hydrocolloid, for example.
- the viscosity of an aqueous composition before the contacting step measured at about 20-25 °C, can be about 0-10000 cPs (or any integer between 0-10000 cPs). Since the aqueous composition can be a hydrocolloid or the like in certain embodiments, it should be apparent that the method can be used to increase the viscosity of aqueous compositions that are already viscous.
- the increase in viscosity can be an increase of at least about 1 %, 10%, 100%, 1000%, 100000%, or 1000000% (or any integer between 1 % and 1000000%), for example, compared to the viscosity of the aqueous composition before the mixing or dissolving step. It should be apparent that very large percent increases in viscosity can be obtained with the disclosed method when the aqueous composition has little to no viscosity before the contacting step.
- the contacting step in a method for increasing the viscosity of an aqueous composition can be performed by mixing or dissolving any poly alpha-1 ,3-1 ,6- glucan ether compound(s) disclosed herein in the aqueous composition by any means known in the art.
- mixing or dissolving can be performed manually or with a machine (e.g., industrial mixer or blender, orbital shaker, stir plate, homogenizer, sonicator, bead mill).
- Mixing or dissolving can comprise a homogenization step in certain embodiments.
- Homogenization (as well as any other type of mixing) can be performed for about 5 to 60, 5 to 30, 10 to 60, 10 to 30, 5 to 15, or 10 to 15 seconds (or any integer between 5 and 60 seconds), or longer periods of time as necessary to mix a poly alpha-1 ,3-1 ,6-glucan ether compound with the aqueous composition.
- a homogenizer can be used at about 5000 to 30000 rpm, 10000 to 30000 rpm, 15000 to 30000 rpm, 15000 to 25000 rpm, or 20000 rpm (or any integer between 5000 and 30000 rpm).
- Hydrocolloids and aqueous solutions disclosed herein prepared using a homogenization step can be termed as homogenized hydrocolloids and aqueous solutions.
- the resulting aqueous composition may be filtered, or may not be filtered.
- an aqueous composition prepared with a homogenization step may or may not be filtered.
- the disclosed invention also concerns a method for producing a poly alpha-1 ,3-1 ,6-glucan ether compound.
- This method comprises: contacting poly alpha-1 ,3-1 ,6-glucan in a reaction under alkaline conditions with at least one etherification agent comprising an organic group, wherein the organic group is etherified to the poly alpha-1 ,3-1 ,6-glucan thereby producing a poly alpha-1 ,3- 1 ,6-glucan ether compound. Further regarding this method:
- the poly alpha-1 , 3-1 , 6-glucan has a weight average degree of polymerization (DP W ) of at least 1000,
- the poly alpha-1 ,3-1 ,6-glucan ether compound has a degree of substitution (DoS) with the organic group of about 0.05 to about 3.0.
- a poly alpha-1 ,3-1 ,6-glucan ether compound produced by this method can optionally be isolated.
- Poly alpha-1 ,3-1 ,6-glucan is contacted in a reaction under alkaline conditions with at least one etherification agent comprising an organic group.
- This step can be performed, for example, by first preparing alkaline conditions by contacting poly alpha-1 ,3-1 ,6-glucan with a solvent and one or more alkali hydroxides to provide a mixture (e.g., slurry) or solution.
- the alkaline conditions of the etherification reaction can thus comprise an alkali hydroxide solution.
- the pH of the alkaline conditions can be at least about 1 1 .0, 1 1 .2, 1 1 .4, 1 1 .6, 1 1 .8, 12.0, 12.2, 12.4, 12.6, 12.8, or 13.0.
- alkali hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and/or
- the concentration of alkali hydroxide in a preparation with poly alpha-1 ,3-1 ,6-glucan and a solvent can be from about 1 -70 wt%, 5-50 wt%, 5-10 wt%, 10-50 wt%, 10-40 wt%, or 10-30 wt% (or any integer between 1 and 70 wt%).
- the concentration of alkali hydroxide such as sodium hydroxide can be at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 wt%.
- An alkali hydroxide used to prepare alkaline conditions may be in a completely aqueous solution or an aqueous solution comprising one or more water-soluble organic solvents such as ethanol or isopropanol.
- an alkali hydroxide can be added as a solid to provide alkaline conditions.
- An organic solvent can be added before or after addition of alkali hydroxide.
- the concentration of an organic solvent (e.g., isopropanol or toluene) in a preparation comprising poly alpha-1 ,3-1 ,6-glucan and an alkali hydroxide can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 wt% (or any integer between 10 and 90 wt%).
- an organic solvent e.g., isopropanol or toluene
- solvents that can dissolve poly alpha-1 ,3-1 ,6-glucan can be used when preparing the etherification reaction.
- solvents include, but are not limited to, lithium chlohde(LiCI)/N,N-dimethyl-acetamide (DMAc),
- Poly alpha-1 ,3-1 ,6-glucan can be contacted with a solvent and one or more alkali hydroxides by mixing. Such mixing can be performed during or after adding these components with each other. Mixing can be performed by manual mixing, mixing using an overhead mixer, using a magnetic stir bar, or shaking, for example.
- poly alpha-1 ,3-1 ,6-glucan can first be mixed in water or an aqueous solution before it is mixed with a solvent and/or alkali hydroxide. After contacting poly alpha-1 ,3-1 ,6-glucan, solvent, and one or more alkali hydroxides with each other, the resulting composition can optionally be
- ambient temperature refers to a temperature between about 15-30 °C or 20-25 °C (or any integer between 15 and 30 °C).
- the composition can be heated with or without reflux at a temperature from about 30 °C to about 150 °C (or any integer between 30 and 150 °C) for up to about 48 hours.
- the composition in certain embodiments can be heated at about 55 °C for about 30 minutes or about 60 minutes.
- composition obtained from mixing a poly alpha-1 ,3-1 ,6-glucan, solvent, and one or more alkali hydroxides with each other can be heated at about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 °C for about 30-90 minutes.
- the resulting composition can optionally be filtered (with or without applying a temperature treatment step).
- filtration can be performed using a funnel, centrifuge, press filter, or any other method and/or equipment known in the art that allows removal of liquids from solids. Though filtration would remove much of the alkali hydroxide, the filtered poly alpha-1 ,3- 1 ,6-glucan would remain alkaline (i.e., mercerized poly alpha-1 ,3-1 ,6-glucan), thereby providing alkaline conditions.
- An etherification agent comprising an organic group is contacted with poly alpha-1 ,3-1 ,6-glucan in a reaction under alkaline conditions in a method herein of producing poly alpha-1 ,3-1 ,6-glucan ether compounds.
- an etherification agent can be added to a composition prepared by contacting poly alpha-1 ,3-1 ,6-glucan, solvent, and one or more alkali hydroxides with each other as described above.
- an etherification agent can be included when preparing the alkaline conditions (e.g., an etherification agent can be mixed with poly alpha-1 , 3-1 , 6-glucan and solvent before mixing with alkali hydroxide).
- An etherification agent herein refers to an agent that can be used to etherify one or more hydroxyl groups of glucose monomeric units of poly alpha- 1 ,3-1 ,6-glucan with an organic group as disclosed herein. Examples of such organic groups include alkyl groups, hydroxy alkyl groups, and carboxy alkyl groups. One or more etherification agents may be used in the reaction.
- Etherification agents suitable for preparing an alkyl poly alpha-1 ,3-1 ,6- glucan ether compound include, for example, dialkyi sulfates, dialkyi carbonates, alkyl halides (e.g., alkyl chloride), iodoalkanes, alkyl inflates (alkyl
- examples of etherification agents for producing methyl poly alpha-1 , 3-1 , 6-glucan ethers include dimethyl sulfate, dimethyl carbonate, methyl chloride, iodomethane, methyl triflate and methyl fluorosulfonate.
- examples of etherification agents for producing ethyl poly alpha-1 ,3-1 ,6-glucan ethers include diethyl sulfate, diethyl carbonate, ethyl chloride, iodoethane, ethyl triflate and ethyl fluorosulfonate.
- Examples of etherification agents for producing propyl poly alpha-1 ,3-1 ,6-glucan ethers include dipropyl sulfate, dipropyl carbonate, propyl chloride, iodopropane, propyl triflate and propyl fluorosulfonate.
- Examples of etherification agents for producing butyl poly alpha-1 ,3-1 ,6-glucan ethers include dibutyl sulfate, dibutyl carbonate, butyl chloride, iodobutane and butyl triflate.
- Etherification agents suitable for preparing a hydroxyalkyl poly alpha-1 , 3- 1 ,6-glucan ether compound include, for example, alkylene oxides such as ethylene oxide, propylene oxide (e.g., 1 ,2-propylene oxide), butylene oxide (e.g., 1 ,2-butylene oxide; 2,3-butylene oxide; 1 ,4-butylene oxide), or combinations thereof.
- propylene oxide can be used as an etherification agent for preparing hydroxypropyl poly alpha-1 ,3-1 ,6-glucan
- ethylene oxide can be used as an etherification agent for preparing hydroxyethyl poly alpha-1 , 3-1 ,6- glucan.
- hydroxyalkyl halides e.g., hydroxyalkyl chloride
- hydroxyalkyl halides can be used as etherification agents for preparing hydroxyalkyl poly alpha-1 ,3-1 ,6- glucan.
- hydroxyalkyl halides include hydroxyethyl halide, hydroxypropyl halide (e.g., 2-hydroxypropyl chloride, 3-hydroxypropyl chloride) and hydroxybutyl halide.
- alkylene chlorohydrins can be used as etherification agents for preparing hydroxyalkyl poly alpha-1 ,3-1 ,6-glucan.
- Alkylene chlorohydrins that can be used include, but are not limited to, ethylene chlorohydrin, propylene chlorohydrin, butylene chlorohydrin, or combinations of these.
- Etherification agents suitable for preparing a dihydroxyalkyl poly alpha- 1 ,3-1 ,6-glucan ether compound include dihydroxyalkyl halides (e.g.,
- dihydroxyalkyl chloride such as dihydroxyethyl halide, dihydroxypropyl halide (e.g., 2,3-dihydroxypropyl chloride [i.e., 3-chloro-1 ,2-propanediol]), or
- dihydroxybutyl halide for example.
- 2,3-dihydroxypropyl chloride can be used to prepare dihydroxypropyl poly alpha-1 ,3-1 ,6-glucan, for example.
- Etherification agents suitable for preparing a carboxyalkyl poly alpha-1 , 3- 1 ,6-glucan ether compound may include haloalkylates (e.g., chloroalkylate).
- haloalkylates examples include haloacetate (e.g., chloroacetate), 3- halopropionate (e.g., 3-chloropropionate) and 4-halobutyrate (e.g., 4- chlorobutyrate).
- chloroacetate e.g., chloroacetate
- 3- halopropionate e.g., 3-chloropropionate
- 4-halobutyrate e.g., 4- chlorobutyrate
- chloroacetate dichloroacetate
- sodium chloroacetate can be used as an etherification agent to prepare carboxymethyl poly alpha-1 , 3-1 , 6-glucan.
- two or more different etherification agents When producing a poly alpha-1 ,3-1 ,6-glucan ether compound with two or more different organic groups, two or more different etherification agents would be used, accordingly.
- both an alkylene oxide and an alkyl chloride could be used as etherification agents to produce an alkyl hydroxyalkyl poly alpha-1 ,3-1 ,6-glucan ether.
- Any of the etherification agents disclosed herein may therefore be combined to produce poly alpha-1 ,3-1 ,6-glucan ether compounds with two or more different organic groups.
- Such two or more etherification agents may be used in the reaction at the same time, or may be used
- any of the temperature- treatment (e.g., heating) steps disclosed below may optionally be used between each addition.
- One may choose sequential introduction of etherification agents in order to control the desired DoS of each organic group.
- a particular etherification agent would be used first if the organic group it forms in the ether product is desired at a higher DoS compared to the DoS of another organic group to be added.
- the amount of etherification agent to be contacted with poly alpha-1 ,3-1 ,6- glucan in a reaction under alkaline conditions can be determined based on the DoS required in the poly alpha-1 ,3-1 ,6-glucan ether compound being produced.
- the amount of ether substitution groups on each glucose monomeric unit in poly alpha-1 ,3-1 ,6-glucan ether compounds produced herein can be determined using nuclear magnetic resonance (NMR) spectroscopy.
- the molar substitution (MS) value for poly alpha-1 ,3-1 ,6-glucan has no upper limit. In general, an
- etherification agent can be used in a quantity of at least about 0.05 mole per mole of poly alpha-1 ,3-1 ,6-glucan. There is no upper limit to the quantity of etherification agent that can be used.
- Reactions for producing poly alpha-1 ,3-1 ,6-glucan ether compounds herein can optionally be carried out in a pressure vessel such as a Parr reactor, an autoclave, a shaker tube or any other pressure vessel well known in the art.
- a pressure vessel such as a Parr reactor, an autoclave, a shaker tube or any other pressure vessel well known in the art.
- a reaction herein can optionally be heated following the step of contacting poly alpha-1 ,3-1 ,6-glucan with an etherification agent under alkaline conditions.
- the reaction temperatures and time of applying such temperatures can be varied within wide limits.
- a reaction can optionally be maintained at ambient temperature for up to 14 days.
- a reaction can be heated, with or without reflux, between about 25 °C to about 200 °C (or any integer between 25 and 200 °C).
- Reaction time can be varied correspondingly: more time at a low temperature and less time at a high temperature.
- a reaction can be heated to about 55 °C for about 3 hours.
- a reaction for preparing a carboxyalkyl poly alpha-1 ,3-1 ,6-glucan herein can be heated to about 50 °C to about 60 °C (or any integer between 50 and 60 °C) for about 2 hours to about 5 hours, for example.
- Etherification agents such as a haloacetate (e.g., monochloroacetate) may be used in these embodiments, for example.
- an etherification reaction herein can be maintained under an inert gas, with or without heating.
- inert gas refers to a gas which does not undergo chemical reactions under a set of given conditions, such as those disclosed for preparing a reaction herein.
- All of the components of the reactions disclosed herein can be mixed together at the same time and brought to the desired reaction temperature, whereupon the temperature is maintained with or without stirring until the desired poly alpha-1 ,3-1 ,6-glucan ether compound is formed.
- the mixed components can be left at ambient temperature as described above.
- Neutralization of a reaction can be performed using one or more acids.
- neutral pH refers to a pH that is neither substantially acidic or basic (e.g., a pH of about 6-8, or about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0).
- acids that can be used for this purpose include, but are not limited to, sulfuric, acetic (e.g., glacial acetic), hydrochloric, nitric, any mineral (inorganic) acid, any organic acid, or any combination of these acids.
- a poly alpha-1 ,3-1 ,6-glucan ether compound produced in a reaction herein can optionally be washed one or more times with a liquid that does not readily dissolve the compound.
- poly alpha-1 ,3-1 ,6-glucan ether can typically be washed with alcohol, acetone, aromatics, or any combination of these, depending on the solubility of the ether compound therein (where lack of solubility is desirable for washing).
- a solvent comprising an organic solvent such as alcohol is preferred for washing a poly alpha-1 ,3-1 ,6-glucan ether.
- a poly alpha-1 ,3-1 ,6-glucan ether product can be washed one or more times with an aqueous solution containing methanol or ethanol, for example. For example, 70-95 wt% ethanol can be used to wash the product.
- a poly alpha-1 ,3- 1 ,6-glucan ether product can be washed with a methanol :acetone (e.g., 60:40) solution in another embodiment.
- a poly alpha-1 ,3-1 ,6-glucan ether produced in the disclosed reaction can be isolated. This step can be performed before or after neutralization and/or washing steps using a funnel, centrifuge, press filter, or any other method or equipment known in the art that allows removal of liquids from solids.
- An isolated poly alpha-1 ,3-1 ,6-glucan ether product can be dried using any method known in the art, such as vacuum drying, air drying, or freeze drying.
- This approach may be suitable for increasing the DoS of an organic group, and/or adding one or more different organic groups to the ether product.
- the structure, molecular weight and DoS of a poly alpha-1 ,3-1 ,6-glucan ether product can be confirmed using various physiochemical analyses known in the art such as NMR spectroscopy and size exclusion chromatography (SEC).
- poly alpha-1 ,3-1 ,6-glucan Any of the embodiments of poly alpha-1 ,3-1 ,6-glucan described above can be used in an etherification reaction herein.
- the poly alpha-1 ,3-1 ,6- glucan used in an etherification reaction herein can be a product of a
- glucosyltransferase enzyme comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the glucosyltransferase enzyme can comprise an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- Poly alpha-1 ,3-1 ,6-glucan used for preparing poly alpha-1 ,3-1 ,6-glucan ether compounds herein can be enzymatically produced from sucrose using one or more glucosyltransferase (gtf) enzymes.
- the poly alpha-1 ,3-1 ,6-glucan product of this enzymatic reaction can be purified before using it to prepare an ether.
- a poly alpha-1 ,3-1 ,6-glucan product of a gtf reaction can be used with little or no processing for preparing poly alpha-1 , 3-1 , 6-glucan ether compounds.
- a poly alpha-1 ,3-1 ,6-glucan slurry can be used directly in any of the above processes for producing a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein.
- a "poly alpha-1 ,3-1 ,6-glucan slurry" refers to a mixture comprising the components of a gtf enzymatic reaction.
- a gtf enzymatic reaction can include, in addition to poly alpha-1 ,3-1 ,6-glucan itself, various components such as sucrose, one or more gtf enzymes, glucose, fructose, leucrose, buffer, FermaSure ® , soluble oligosaccharides, oligosaccharide primers, bacterial enzyme extract components, borates, sodium hydroxide, hydrochloric acid, cell lysate, proteins and/or nucleic acids.
- the components of a gtf enzymatic reaction can include, in addition to poly alpha-1 , 3-1 , 6-glucan itself, sucrose, one or more gtf enzymes, glucose and fructose, for example.
- the components of a gtf enzymatic reaction can include, in addition to poly alpha-1 ,3-1 ,6-glucan itself, sucrose, one or more gtf enzymes, glucose, fructose, leucrose and soluble oligosaccharides (and optionally bacterial enzyme extract components). It should be apparent that poly alpha-1 ,3-1 ,6- glucan, when in a slurry as disclosed herein, has not been purified or washed.
- a slurry represents a gtf enzymatic reaction that is complete or for which an observable amount of poly alpha-1 ,3-1 ,6-glucan has been produced, which forms a solid since it is insoluble in the aqueous reaction milieu (pH of 5-7, for example).
- a poly alpha-1 ,3-1 ,6-glucan slurry can be prepared by setting up a gtf reaction as disclosed herein.
- a wet cake of poly alpha-1 ,3-1 ,6-glucan can be used directly in any of the above processes for producing a poly alpha-1 ,3-1 ,6-glucan ether compound disclosed herein.
- a "wet cake of poly alpha-1 ,3-1 ,6-glucan" as used herein refers to poly alpha-1 ,3-1 ,6-glucan that has been separated (e.g., filtered) from a slurry and washed with water or an aqueous solution.
- a wet cake can be washed at least 1 , 2, 3, 4, 5, or more times, for example.
- the poly alpha-1 ,3-1 ,6- glucan is not dried when preparing a wet cake.
- a wet cake is termed as "wet” given the retention of water by the washed poly alpha-1 ,3-1 ,6-glucan.
- a wet cake of poly alpha-1 ,3-1 ,6-glucan can be prepared using any device known in the art for separating solids from liquids, such as a filter or centrifuge.
- a filter or centrifuge For example, poly alpha-1 ,3-1 , 6-glucan solids in a slurry can be collected on a funnel using a mesh screen over filter paper. Filtered wet cake can be
- poly alpha-1 ,3-1 ,6-glucan solids from a slurry can be collected as a pellet via centrifugation, resuspended in water (e.g., deionized water), and re-pelleted and resuspended one or more additional times.
- compositions and methods disclosed herein include:
- a reaction solution comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1 ,3-1 ,6-glucan, wherein the
- glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- the glucan has a weight average degree of polymerization (DP W ) of at least 1000.
- glucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
- a method for producing poly alpha-1 ,3-1 ,6-glucan comprising:
- glucosyltransferase enzyme that synthesizes poly alpha-1 ,3-1 ,6-glucan, wherein the glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10;
- step (b) optionally, isolating the poly alpha-1 ,3-1 ,6-glucan produced in step (a).
- the glucan has a weight average degree of polymerization (DP W ) of at least 1000.
- the disclosed invention is further defined in Examples 1 -8 provided below.
- T10 dextran (D9260), IPTG, (cat#l6758), triphenyltetrazolium chloride, and BCA protein assay kit/reagents were obtained from the Sigma Co. (St. Louis, MO).
- BELLCO spin flasks were from the Bellco Co. (Vineland, NJ).
- LB medium was from Becton, Dickinson and Company (Franklin Lakes, NJ).
- Suppressor 7153 antifoam was obtained from Cognis Corporation (Cincinnati, OH). All other chemicals were obtained from commonly used suppliers of such chemicals.
- the seed medium used to grow starter cultures for the fermenters contained: yeast extract (AMBEREX 695, 5.0 grams per liter, g/L), K 2 HPO 4 (10.0 g/L), KH 2 PO 4 (7.0 g/L), sodium citrate dihydrate (1 .0 g/L), (NH 4 ) 2 SO 4 (4.0 g/L), MgSO 4 heptahydrate (1 .0 g/L) and ferric ammonium citrate (0.10 g/L).
- the pH of the medium was adjusted to 6.8 using either 5N NaOH or H 2 SO and the medium was sterilized in the flask. Post-sterilization additions included glucose (20 mL/L of a 50% w/w solution) and ampicillin (4 mL/L of a 25 mg/mL stock solution).
- Fermenter Medium included: glucose (20 mL/L of a 50% w/w solution) and ampicillin (4 mL/L of a 25 mg/mL stock solution).
- the growth medium used in the fermenter contained: KH 2 PO (3.50 g/L), FeSO 4 heptahydrate (0.05 g/L), MgSO 4 heptahydrate (2.0 g/L), sodium citrate dihydrate (1 .90 g/L), yeast extract (AMBEREX 695, 5.0 g/L), Suppressor 7153 antifoam (0.25 mL/L), NaCI (1 .0 g/L), CaCI 2 dihydrate (10 g/L), and NIT trace elements solution (10 mL/L).
- the NIT trace elements solution contained citric acid monohydrate (10 g/L), MnSO 4 hydrate (2 g/L), NaCI (2 g/L), FeSO 4 heptahydrate (0.5 g/L), ZnSO 4 heptahydrate (0.2 g/L), CuSO 4 pentahydrate (0.02 g/L) and NaMoO 4 dihydrate (0.02 g/L).
- Post-sterilization additions included glucose (12.5 g/L of a 50% w/w solution) and ampicillin (4 mL/L of a 25 img/mL stock solution).
- Production of a recombinant gtf enzyme in a fermenter was initiated by preparing a pre-seed culture of an E. coli strain expressing the gtf enzyme. A 10- mL aliquot of seed medium was added into a 125-mL disposable baffled flask and inoculated with a 1 .0-mL aliquot of the E. coli strain in 20% glycerol. The culture was allowed to grow at 37 °C while shaking at 300 rpm for 3 hours.
- a seed culture which was used for starting growth for gtf fermentation, was prepared by charging a 2-L shake flask with 0.5 L of seed medium. 1 .0 mL of the pre-seed culture was aseptically transferred into 0.5-L seed medium in the flask and cultivated at 37 °C and 300 rpm for 5 hours. The seed culture was transferred at an optical density 550 nm (OD 550 ) > 2 to a 14-L fermenter (Braun, Perth Amboy, NJ) containing 8 L of fermenter medium at 37 °C.
- the E. coli strain was allowed to grow in the fermenter medium.
- Glucose 50% w/w glucose solution containing 1 % w/w MgSO 4 7H 2 O
- the glucose feed was started at 0.36 grams feed per minute (g feed/min) and increased
- the DO was controlled first by impeller agitation rate (400 to 1200 rpm) and later by aeration rate (2 to 10 standard liters per minute, slpm).
- Culture pH was controlled at 6.8 using NH 4 OH (14.5% w/v) and H 2 SO 4 (20% w/v). Back pressure was maintained at 0.5 bars.
- 5 ml_ of Suppressor 7153 antifoam was added to the fermenter to suppress foaming.
- Cells were harvested by centrifugation 8 hours post IPTG addition and were stored at -80 °C as a cell paste.
- the cell paste obtained from fermentation for each gtf enzyme was suspended at 150 g/L in 50 mM potassium phosphate buffer, pH 7.2, to prepare a slurry.
- the slurry was homogenized at 12,000 psi (Rannie-type machine, APV- 1000 or APV 16.56) and the homogenate chilled to 4 °C.
- 50 g of a floe solution (Sigma Aldrich no. 409138, 5% in 50 mM sodium phosphate buffer, pH 7.0) was added per liter of cell homogenate.
- Gtf enzyme activity was confirmed by measuring the production of reducing sugars (fructose and glucose) in a gtf reaction solution.
- a reaction solution was prepared by adding a gtf extract (prepared as above) to a mixture containing sucrose (50 g/L), potassium phosphate buffer (pH 6.5, 50 mM), and dextran T10 (1 mg/nnL); the gtf extract was added to 5% by volume. The reaction solution was then incubated at 22-25 °C for 24-30 hours, after which it was centrifuged. Supernatant (0.01 mL) was added to a mixture containing 1 N NaOH and 0.1 % triphenyltetrazolium chloride (Sigma-Aldrich). The mixture was incubated for five minutes after which its OD 48 onm was determined using an ULTROSPEC spectrophotometer (Pharmacia LKB, New York, NY) to gauge the presence of the reducing sugars fructose and glucose.
- Glycosidic linkages in glucan products synthesized by a gtf enzyme were determined by 13 C NMR (nuclear magnetic resonance) or 1 H NMR.
- 13 C NMR dry glucan polymer (25-30 mg) was dissolved in 1 ml_ of deuterated DMSO containing 3% by weight of LiCI with stirring at 50 °C. Using a glass pipet, 0.8 ml_ of the solution was transferred into a 5-mm NMR tube.
- a quantitative 13 C NMR spectrum was acquired using a Bruker Avance 500-MHz NMR spectrometer (Billerica, MA) equipped with a CPDUL cryoprobe at a spectral frequency of 125.76 MHz, using a spectral window of 26041 .7 Hz.
- An inverse-gated decoupling pulse sequence using waltz decoupling was used with an acquisition time of 0.629 second, an inter-pulse delay of 5 seconds, and 6000 pulses.
- the time domain data was transformed using an exponential
- a glucan polymer sample was weighed into a vial on an analytical balance.
- the vial was removed from the balance and 0.8 ml_ of deuterated DMSO (DMSO-d6), containing 3% by weight of LiCI, was added to the vial.
- DMSO-d6 deuterated DMSO
- the mixture was stirred with a magnetic stir bar and warmed to 90 °C until the glucan sample dissolved.
- the solution was allowed to cool to room temperature. While stirring at room temperature, 0.2 mL of a 20% by volume solution of trifluoroacetic acid (TFA) in DMSO-d6 was added to the polymer solution.
- TFA trifluoroacetic acid
- the TFA was added in order to move all hydroxyl proton signals out of the region of the spectrum where carbohydrate ring proton signals occur.
- a portion, 0.8 mL, of the final solution was transferred, using a glass pipet, into a 5-mm NMR tube.
- a quantitative 1 H NMR spectrum was acquired using an NMR spectrometer with a proton frequency of 500 MHz or greater.
- the spectrum was acquired using a spectral window of 1 1 .0 ppm and a transmitter offset of 5.5 ppm.
- a 90° pulse was applied for 32 pulses with an inter-pulse delay of 10 seconds and an acquisition time of 1 .5 seconds.
- the time domain data were transformed using an exponential multiplication of 0.15 Hz.
- the DPw of a glucan product synthesized by a gtf enzyme was determined by SEC. Dry glucan polymer was dissolved in DMAc and 5% LiCI (0.5 mg/mL) with shaking overnight at 100 °C.
- the SEC system used was an AllianceTM 2695 separation module from Waters Corporation (Milford, MA) coupled with three on- line detectors: a differential refractometer 2410 from Waters, a multiangle light scattering photometer HeleosTM 8+ from Wyatt Technologies (Santa Barbara, CA), and a differential capillary viscometer ViscoStarTM from Wyatt.
- the columns used for SEC were four styrene-divinyl benzene columns from Shodex (Japan) and two linear KD-806M, KD-802 and KD-801 columns to improve resolution at the low molecular weight region of a polymer distribution.
- the mobile phase was DMAc with 0.1 1 % LiCI.
- the chromatographic conditions used were 50 °C in the column and detector compartments, 40 °C in the sample and injector
- EmpowerTM version 3 from Waters (calibration with broad glucan polymer standard) and Astra ® version 6 from Wyatt (triple detection method with column calibration).
- This Example describes preparing an N-terminally truncated version of a
- Streptococcus oralis gtf enzyme identified in GENBANK under Gl number 7684297 (SEQ ID NO:2, encoded by SEQ ID NO:1 ; herein referred to as "4297").
- a nucleotide sequence encoding gtf 4297 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc., Menlo Park, CA).
- This plasmid construct was used to transform E. coli MG1655 (ATCCTM 47076) cells to generate the strain identified as
- This Example describes preparing an N-terminally truncated version of a Streptococcus sp. C150 gtf enzyme identified in GENBANK under Gl number 322373298 (SEQ ID NO:4, encoded by SEQ ID NO:3; herein referred to as "3298").
- a nucleotide sequence encoding gtf 3298 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.).
- the nucleic acid product (SEQ ID NO:3), encoding gtf 3298 (SEQ ID NO:4), was subcloned into pJexpress404 ® to generate the plasmid construct identified as pMP98.
- This plasmid construct was used to transform E. coli MG1655 (ATCCTM 47076) cells to generate the strain identified as MG1655/pMP98.
- This Example describes preparing an N-terminally truncated version of a Streptococcus mutans gtf enzyme identified in GENBANK under Gl number 290580544 (SEQ ID NO:6, encoded by SEQ ID NO:5; herein referred to as "0544").
- a nucleotide sequence encoding gtf 0544 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.).
- This plasmid construct was used to transform E. coli MG1655 (ATCCTM 47076) cells to generate the strain identified as MG1655/pMP67.
- This Example describes preparing an N-terminally truncated version of a
- Streptococcus sanguinis gtf enzyme identified in GENBANK under Gl number 328945618 SEQ ID NO:8, encoded by SEQ ID NO:7; herein referred to as "5618").
- a nucleotide sequence encoding gtf 5618 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.).
- the nucleic acid product (SEQ ID NO:7), encoding gtf 5618 (SEQ ID NO:8), was subcloned into pJexpress404 ® to generate the plasmid construct identified as pMP72.
- This plasmid construct was used to transform E. coli MG1655 (ATCCTM 47076) cells to generate the strain identified as MG1655/pMP72.
- This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under Gl number 662379 (SEQ ID NO:10, encoded by SEQ ID NO:9; herein referred to as "2379").
- a nucleotide sequence encoding gtf 2379 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.).
- the nucleic acid product (SEQ ID NO:9), encoding gtf 2379 (SEQ ID NO:10), was subcloned into pJexpress404 ® to generate the plasmid construct identified as pMP65.
- This plasmid construct was used to transform E. coli MG1655 (ATCCTM 47076) cells to generate the strain identified as MG1655/pMP65.
- gtf reaction solutions were prepared comprising sucrose (50 g/L), potassium phosphate buffer (pH 6.5, 50 mM) and a gtf enzyme (2.5% extract by volume). After 24-30 hours at 22-25 °C, insoluble glucan polymer product was harvested by centrifugation, washed three times with water, washed once with ethanol, and dried at 50 °C for 24-30 hours.
- gtf enzymes capable of producing insoluble glucan polymer having a heterogeneous glycosidic linkage profile (alpha-1 ,3 and 1 ,6 linkages) and a DPw of at least 1000 were identified. These enzymes can be used to produce insoluble poly alpha-1 ,3-1 ,6-glucan suitable for derivatization to downstream products such as glucan ether, as demonstrated below in Example 7.
- Poly alpha-1 ,3-1 ,6-glucan was first prepared as in Example 6, but with a few modifications. Specifically, a glucan polymerization reaction solution was prepared comprising sucrose (300 g), potassium phosphate buffer (pH 5.5; 8.17 g), gtf enzyme 4297 extract (90 mL) in 3 L distilled water. After 24-30 hours at 22-25 °C, insoluble glucan polymer was harvested by centrifugation, filtered, washed three times with water, washed twice with ethanol, and dried at 50 °C for 24-30 hours. About 12 g of poly alpha-1 ,3-1 ,6-glucan was obtained.
- the DPw and glycosidic linkages of the insoluble glucan polymer was determined as described in the General Methods.
- the polymer had a DPw of 10,540 and a linkage profile of 31 % alpha-1 ,3 and 67% alpha-1 ,6. It had a weight-average molecular weight (M w ) of 1 100000.
- M w weight-average molecular weight
- the solid material was then collected by vacuum filtration and washed with ethanol (70%) four times, dried under vacuum at 20-25 °C, and analyzed by NMR to determine degree of substitution (DoS) of the solid.
- DoS degree of substitution
- the solid was identified as sodium carboxymethyl poly alpha-1 ,3-1 ,6-glucan with a DoS of 0.464 (sample 1 D in Table 3).
- Table 3 provides a list of DoS measurements for additional samples of carboxymethyl poly alpha-1 ,3-1 ,6-glucan prepared using processes similar to the above process, but with certain modifications as indicated in the table.
- Each reaction listed in Table 3 used poly alpha-1 ,3-1 ,6-glucan with an M w of 1 100000 as substrate. The results in Table 3 indicate that by altering the reagent amounts and time of the etherification reaction, product DoS can be altered.
- a Reagent refers to sodium monochloroacetate.
- glucan ether derivative carboxymethyl poly alpha-1 ,3-1 ,6- glucan
- This Example describes the effect of carboxymethyl poly alpha-1 ,3-1 ,6- glucan on the viscosity of an aqueous composition.
- Example 72 Various sodium carboxymethyl poly alpha-1 ,3-1 ,6 glucan samples (1A-1 D) were prepared as described in Example 72. To prepare 0.6 wt% solutions of each of these samples, 0.102 g of sodium carboxymethyl poly alpha-1 ,3-1 ,6- glucan was added to Dl water (17 g). Each preparation was then mixed using a bench top vortexer at 1000 rpm until the solid was completely dissolved.
- This Example describes producing carboxymethyl dextran for use in Example 10.
- Sodium hydroxide (0.9 mL of a 15% solution) was added dropwise to the preparation, which was then heated to 25 °C on a hotplate. The preparation was stirred for 1 hour before the temperature was increased to 55 °C.
- a Reagent refers to sodium monochloroacetate.
- This Example describes the viscosity, and the effect of shear rate on viscosity, of solutions containing the carboxymethyl dextran samples prepared in Example 9.
- Various sodium carboxymethyl dextran samples (2A and 2B) were prepared as described in Example 9.
- carboxymethyl poly alpha-1 ,3-1 ,6-glucan samples at the same low concentration (0.6 wt%) in water.
- Table 4 indicates that carboxymethyl poly alpha- 1 ,3-1 ,6-glucan solutions have viscosities of about 48-2010 cPs.
- carboxymethyl dextran sample 2B which likely has a higher molecular weight than the molecular weights of the carboxymethyl poly alpha-1 ,3-1 ,6-glucan samples.
- carboxymethyl dextran sample 2B Despite having a higher molecular weight, carboxymethyl dextran sample 2B exhibited a substantially lower viscosity-modifying effect than carboxymethyl poly alpha-1 ,3-1 ,6-glucan.
- carboxymethyl poly alpha-1 ,3-1 ,6-glucan has a greater viscosity-modifying effect than carboxymethyl dextran.
- This Example describes producing carboxymethyl poly alpha-1 , 3-glucan for use in Example 12.
- Poly alpha-1 , 3-glucan was prepared using a gtfJ enzyme preparation as described in U.S. Patent Appl. Publ. No. 2013/0244288, which is incorporated herein by reference in its entirety.
- the solid material was then collected by vacuum filtration and washed with ethanol (70%) four times, dried under vacuum at 20-25 °C, and analyzed by NMR to determine degree of substitution (DoS) of the solid.
- DoS degree of substitution
- a Reagent refers to sodium monochloroacetate.
- This Example describes the effect of carboxymethyl poly alpha-1 ,3-glucan on the viscosity of an aqueous composition.
- Example 1 1 Various sodium carboxymethyl poly alpha-1 ,3-glucan samples (C1A and C1 B) were prepared as described in Example 1 1 . To prepare 0.6 wt% solutions of each of these samples, 0.102 g of sodium carboxymethyl poly alpha-1 ,3- glucan was added to Dl water (17 g). Each preparation was then mixed using a bench top vortexer at 1000 rpm until the solid was completely dissolved.
- carboxymethyl poly alpha-1 ,3-1 , 6-glucan may have a greater viscosity-modifying effect than carboxymethyl poly alpha-1 ,3- glucan.
- CMC samples (C3A and C3B, Table 9) obtained from DuPont Nutrition & Health (Danisco) were dissolved in Dl water to prepare 0.6 wt% solutions of each sample.
- CMC (0.6 wt%) therefore can increase the viscosity of an aqueous solution. However, it is believed that this ability to increase viscosity is lower than the ability of carboxymethyl poly alpha-1 ,3-1 ,6-glucan to increase viscosity.
- carboxymethyl poly alpha-1 ,3-1 ,6-glucan may have a greater viscosity-modifying effect than CMC.
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US10106626B2 (en) | 2014-01-17 | 2018-10-23 | Ei Du Pont De Nemours And Company | Production of poly alpha-1,3-glucan formate films |
US9926541B2 (en) * | 2014-02-14 | 2018-03-27 | E I Du Pont De Nemours And Company | Glucosyltransferase enzymes for production of glucan polymers |
PL3122887T3 (en) | 2014-03-25 | 2018-11-30 | E. I. Du Pont De Nemours And Company | Production of glucan polymers from unrefined sucrose |
WO2015200612A1 (en) * | 2014-06-26 | 2015-12-30 | E. I. Du Pont De Nemours And Company | Production of poly alpha-1,3-glucan food casings |
EP3237455B1 (en) | 2014-12-22 | 2020-03-04 | DuPont Industrial Biosciences USA, LLC | Polysaccharide compositions for absorbing aqueous liquid |
CN107406524B (en) | 2014-12-22 | 2020-12-08 | 杜邦工业生物科学美国有限责任公司 | Polymer blends containing poly alpha-1, 3-glucan |
ES2803024T3 (en) | 2015-02-06 | 2021-01-22 | Dupont Ind Biosciences Usa Llc | Colloidal dispersions of poly-alpha-1,3-glucan-based polymers |
EP3277730B1 (en) * | 2015-04-03 | 2022-02-09 | Nutrition & Biosciences USA 4, Inc. | Gelling dextran ethers |
CN107404924B (en) | 2015-04-03 | 2022-07-19 | 营养与生物科学美国4公司 | Oxidized dextran |
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